The virulence of Shigella flexneri, etiologic agent of bacillary dysentery, requires the use of a type III secretion system (TTSS) to deliver IpaB and IpaC to target cell membranes creating a pore for passage of other proteins into the host cytoplasm to promote bacterial entry. IpaD is also required for invasion but its precise role in unknown. Deletion mutagenesis shows that IpaD controls the proper secretion and membrane insertion of IpaB/lpaC, while microscopy shows that IpaD resides at the exposed TTSS needle tip. Thus, we hypothesize that IpaD functions from the tip of the TTSS needle to control the mobilization of IpaB to the needle tip, which ultimately controls the insertion of IpaB and IpaC into the host cell membrane. Molecular dissection, structural analysis, biophysical characterization, and microscopic imaging are expected to reveal how IpaD is positioned at the needle tip and how it triggers the secretion of IpaB to the needle tip. Therefore, the specific aims of this investigation are to: 1) Establish the position of IpaD within the TTSS pre- and post-secretion stimulus and define the molecular basis for its control of IpaB recruitment to the top complex. Mutagenesis will be used to ascertain the roles of the IpaD domains in tip localization and IpaB mobilization. Electron microscopy will be used to determine the position of the domains and image the ternary complex structure at the needle tip. 2) Determine the basis for interactions between multiple IpaDs and IpaB within the needle tip complex. Biophysical analyses will be used to assess the IpaD oligomerization state and determine what molecular contacts are required for this oligomerization. Fluorescence spectroscopy will then be used to analyze the interaction between IpaB and IpaD. 3) Determine how IpaD interacts with MxiH by NMR spectroscopy. It is proposed that the C-terminal coil of IpaD interacts with MxiH. Therefore, NMR can be used to assess this interaction and detect the residues of MxiH that interact with IpaD, which will aid in docking the IpaD to MxiH in molecular simulations. This tightly focused, interdisciplinary investigation targets IpaD, a protein required for Shigella infection. Because the initial mechanism of invasion is conserved among a broad range of gram-negative bacteria, the completion of this study will have long term implications in understanding how this set of diverse gram-negative bacteria set-up infection. The understanding how IpaD mobilizes IpaB is expected to lead to the development of new vaccines and drugs for preventing dysentery.